Summary of work: The transcription factor C/EBPalpha is a positive modulator of transcription for several adipocyte-specific genes which play a role in energy metabolism. These include the insulin-responsive glucose transporter GLUT4, lipid-synthesizing enzymes and leptin, an hormone that acts centrally to regulate body weight through the control of appetite and energy expenditure. To this date, there is little information available regarding regulation of C/EBPalpha expression by metabolic signals. In this study, we examined the effect of glucose, glucosamine, a product of the hexosamine pathway, and xylitol, an activator of the pentose phosphate pathway, on C/EBPalpha gene expression. Addition of glucose resulted in a dose-dependent inhibition of C/EBPalpha mRNA and protein levels in insulin-treated 3T3-L1 adipocytes while having no significant effect on C/EBPbeta mRNA levels. Increased flux through the hexosamine pathway, either by glucosamine or xylitol, mimicked high glucose?s ability to reduce C/EBPalpha mRNA expression. Activation of the hexosamine pathway is known to increase intracellular levels of substrates for protein glycosylation. We found that the control of C/EBPalpha mRNA expression was independent of protein glycosylation. Using a cell line stably transfected with the C/EBPalpha promoter fused to a reporter gene, we demonstrated the presence of regulatory element(s) within this promoter that is/are influenced by metabolic signals. Of interest, treatment of rats with glucose or glucosamine led to a reduction in C/EBPalpha mRNA levels in epididymal fat, but not in omental adipose tissue. Taken together, these results suggest that metabolic signals modulate C/EBPalpha mRNA levels in part through an effect on the C/EBPalpha promoter. We plan to determine the impact such depot-specific regulation of C/EBPalpha gene will have on the control of regional adiposity and fat distribution, and the overall control of energy homeostasis. A manuscript has been submitted to J. Clin. Invest.